Mammalian cells are the preferred host cells for the production of complex biopharmaceutical proteins as the modifications carried out post-translationally are compatible with humans both functionally and pharmacokinetically. Commercially relevant cell types are hybridoma, myeloma CHO (Chinese Hamster Ovary) cells and BHK (Baby Hamster Kidney) cells. The cultivation of the host cells is increasingly carried out under serum- and protein-free production conditions. The reasons for these are the concomitant cost reduction, the reduced interference in the purification of the recombinant protein and the reduction in the potential for the introduction of pathogens (e.g. prions and viruses). The use of CHO cells as host cells is becoming more widespread as these cells adapt to suspension growth in serum- and protein-free medium and are also regarded and accepted as safe production cells by the regulatory authorities.
In order to produce a stable mammalian cell line which expresses a heterologous gene of interest, the heterologous gene is generally inserted in the desired cell line together with a selectable marker gene such as e.g. neomycin phosphotransferase by transfection. The heterologous gene and the selectable marker gene can be expressed either together by a single vector or by separate vectors which are cotransfected. Two to three days after transfection the cells are transferred into medium containing a selective agent, e.g. G418 when using neomycin phosphotransferase-gene, and cultivated for some weeks under these selective conditions. The emergent resistance cells can then be isolated and investigated for expression of the desired gene product. As a result of the random and undirected integration into the host cell genome a population of cells is obtained which have completely different rates of expression of the heterologous gene. These may also include non-expressing cells in which the selectable marker is expressed but not the gene of interest. In order to identify cell clones which have a very high expression of the heterologous gene of interest, it is therefore necessary to examine and test a large number of clones, which is time consuming, labour intensive and expensive.
Gene amplification is a widespread phenomenon in animal cell cultures, which is used for the production of recombinant biopharmaceutical proteins. The gene amplification drastically improves the originally relatively low productivity of numerous mammalian cell lines. One amplification technique which is widely used is dihydrofolate reductase (DHFR)-based gene amplification system which is very often used in DHFR-deficient Chinese Hamster Ovary (CHO) cells. The DHFR-deficient CHO cells, e.g. CHO-DUKX (ATCC CRL-9096) or CHO-DG44 (Urlaub, G. et al., Cell 1983, 33, 405-412), are transfected with a suitable vector system which codes for DHFR and the protein of interest. Then the transfectants are selected in a medium without glycine, hypoxanthine and thymidine. The amplification and hence the establishment of highly productive cell lines is achieved by the increasing addition of methotrexate (MTX), an inhibitor of dihydrofolate reductase (Kaufman, R. J. et al., J Mol Biol 1982, 159, 601-621; U.S. Pat. No. 4,656,134). The subsequent selection of the highly productive cells obtained is also subject to the principle of chance and is based on probabilities, as a result of which this selection step is highly labour-intensive and time-consuming.
All kinds of methods have been developed for monitoring gene transformation and expression better and more rapidly. These include, first of all, the use of reporter molecules such as chloramphenicol-acetyltransferase, luciferase, β-galactosidase or fusion proteins which contain the coding regions of β-galactosidase or luciferase. The disadvantage of these reporter gene assays is that the cells have to be fixed or lysed and have to be incubated with exogenously added substrates and co-factors. Thus, further cultivation of the analysed cells is out of the question. A more recent method based on the co-expression of the E.coli enzyme β-galactosidase does indeed allow lysed cells to be sorted using a FACS apparatus (Nolan, G. P. et al., Proc Natl Acad Sci USA 1988, 85, 2603-2607), but hypotonic pretreatment is required in order to charge the cells with the fluorogenic substrate. This activity also has to be inhibited before the FACS-based sorting.
With the introduction of green fluorescent protein (GFP) from Aequorea victoria and the GFP mutants developed therefrom as reporter molecule it became much easier to identify cells which express a heterologous gene. Co-expression of GFP allowed real-time analysis in vivo and sorting of transfectants on the basis of their fluorescence without the need for additional substrates or co-factors. The use of GFP as a reporter molecule for monitoring gene transfer has been described in various publications. In U.S. Pat. Nos. 5,491,084 and 6,146,826, Chalfie et al. described a method of selecting cells which express a protein of interest. This method comprises co-transfection of cells by a DNA molecule which contains the coding sequence for the protein of interest, and a second DNA-molecule which codes the GFP-gene. Then the GFP-expressing cells are selected. Gubin et al. investigated the stability of GFP expression in CHO cells in the absence of selective growth conditions (Gubin, A. N. et al., Biochem Biophys Res Commun 1997, 236, 347-350). The cells were transfected with a plasmid which contained both GFP and neomycin phosphotransferase. Mosser et al. used a plasmid which contained a bicistronic expression cassette coding for a GFP and a target gene (also known as the gene of interest) to identify and select cells which expressed inducible product (Mosser, D. D. et al., BioTechniques 1997, 22, 150-161). The target gene was under the control of a regulatable promoter. The coupling of the GFP and target gene expression was achieved using a viral IRES (Internal Ribosome Entry Site) element, as a result of which a bicistronic mRNA which coded for GFP and the protein of interest was expressed. The plasmid used did not itself contain any selectable marker gene. This was therefore introduced by a second plasmid in a co-transfection or in a subsequent transfection. By contrast, Levenson et al. used retroviral vectors with a bicistronic expression cassette in which the gene of interest can be cloned in front of the IRES sequence (Levenson, V. V. et al., Human Gene Therapy 1998, 9, 1233-1236). The sequence following the IRES sequence, on the other hand, coded for a selectable marker gene, this being a marker which conferred resistance to G418, puromycin, hygromycin B, histidinol D or phleomycin, or it was GFP.
Vectors have already also been described which contain an IRES element from the family of the picorna viruses, the IRES element being positioned between the product gene and a selectable marker gene (Pelletier, J. et al., Nature 1988, 334, 320-325; Jang, S. K. et al., J Virol 1989, 63, 1651-1660; Davies, M. V. et al., J Virol 1992, 66, 1924-1932).
GFP has also been successfully fused with resistance marker genes. For example, Bennett et al. describe a GFP/zeomycin fusion protein (Bennett, R. P. et al., BioTechniques 1998, 24, 478-482). This bifunctional selectable marker was successfully used to identify and select transfected mammalian cells. Primig et al. on the other hand used a fusion protein of GFP and neomycin phosphotransferase for their enhancer studies (Primig, M. et al., Gene 1998, 215, 181-189).
In the publication by Meng et al. and in International Patent application WO 01/04306, an expression system in which the gene of interest was expressed together with the amplifiable selectable marker gene DHFR and a GFP gene from a single vector was used to select and identify cells with a high expression of a recombinant protein (Meng, Y. G. et al., Gene 2000, 242, 201-207). The three genes were either combined in one transcription unit or divided between two units. This spatial and transcriptional linking of all three genes in a single expression vector was intended to increase their probability of co-amplification under selection pressure and thus identify and select high producing clones. The best clones which were isolated by using the combined selection by means of amplifiable DHFR selection markers and GFP-based FACS sorting expressed the protein of interest in an order of magnitude of not more than 3 to 4.5 pg per cell per day. The experiments were carried out with adherent cells and in serum-containing medium, i.e. with cells and under conditions which are known to be substantially more robust and are characterised by higher basic productivities.